The gram positive bacillus Listeria monocytogenes causes bacteremia and meningitis, while the gram negative bacillus Shigella flexneri causes severe diarrhea and dehydration. The pathogenesis of Listeriosis and Shigellosis absolutely requires these intracellular bacteria to usurp the host cell's contractile system to form membrane projections, called filopodia. Filopodia push into adjacent cells where they are ingested, allowing efficient cell-to-cell to spread and avoidance of the humoral immune system, as well as many antibiotics. The actin regulatory proteins and pathways that mediate bacteria-induced filopodia formation are poorly understood. We will: Aim I - Explore the relative contribution of known actin-regulatory proteins to Listeria- and Shigella-induced filopodia formation. Immuno-fluorescence and immuno-gold electron microscopy, expression of GFP fusion proteins, RNAi, and knockout cell lines will be combined with freeze fracture electron microscopy, fluorescence recovery after photo-bleaching (FRAP), and laser nano-scissors to assess the roles of the actin-regulatory proteins Eps8, VASP , Arp2/3 complex, fascin, and myosin-X in filopodia formation. Aim II - Dissect the signal transduction pathways that initiate Listeria-induced filopodia formation. The PI3K inhibitor LY294002 blocks Listeria-induced filopodia formation, and delivery of PIP3 results in a > 6 x increase in filopodia number without a significant change in Listeria intracellular motility. Using targeted RNAi knockdown, the specific PI3K isotype responsible for initiating filopodia formation will be determined. The role of the downstream affecters Akt, PKC-gamma, Tapp-1 and Plekstrin-2, GAPs, GEFs, and small G-proteins will be examined. Filopodia formation in response to changes in [Ca2+]i is being assessed by Fura-2 and BAPTAM, and we find a > 2x increase in filopodia length following Ca2+ chelation. The effects of polyamine calcium channel agonist and antagonists on Listeria cell-to-cell spread will be explored. These investigations promise to clarify the key regulatory pathways required for Listeria- and Shigella-induced filopodia formation and cell-to-cell spread, critical steps for virulence and new therapeutic approaches. These discoveries can be applied to all forms of cell motility, including tumor metastasis, neurite outgrowth, and platelet spreading.